Effects of strength training on osteogenic differentiation and bone strength in aging female Wistar rats

The effects of strength training (ST) on the mechanical bone strength and osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs) from adult, aged and exercised aged rats were determined. The exercised aged animals displayed higher values of areal bone mineral density, compression test, alkaline phosphatase activity (ALP) and biological mineralization, while oil red O staining for adipocytes was lower. ST increased gene expression of runt-related transcription factor 2 (Runx2), osterix (Osx) as well as bone matrix protein expression, and reduced expression of peroxisome proliferator-activated receptor gamma (Pparγ). The production of pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) was lower in BMSCs of the aged exercised group. The ST practice was able to improve the bone mechanical properties in aged female rats, increasing the potential for osteogenic differentiation of BMSCs, reducing the adipogenic differentiation and pro-inflammatory cytokine level. In summary, the data achieved in this study showed that strength training triggers physiological responses that result in changes in the bone microenvironment and bring benefits to biomechanical parameters of bone tissue, which could reduce the risk of fractures during senescent.

Osteoporotic fracture is considered a major worldwide epidemic, resulting in serious increased morbidity and mortality in older women 1,2 . In this phase of life, there is a negative balance between the volumes of bone resorbed and formed during remodeling, which accelerates bone mass loss, leading to architectural deterioration and decreased bone strength 1 . Physical exercise practice, combined or not with pharmacological treatment, has been shown to prevent osteoporosis and osteoporotic fractures in postmenopausal women 2 and aged female rats 3 .
The etiopathogenesis of aging-associated osteoporosis has been linked to changes in the number of mesenchymal stem cells, capacity for osteogenic differentiation 4 and increased pro-inflammatory cytokine levels in bone marrow 5 . It leads to a shift in the bone microenvironment resulting in a decrease in bone mass with increasing age. In addition, physical inactivity in older individuals contributes to increase the differentiation potential of the adipocyte lineage, decreasing bone quality 4 .
Mechanical stimulation of the bone is able to induce differentiation of mechanosensitive mesenchymal stem cells into osteoblasts 6,7 , which in turn activates the transcription factor for regulating osteogenic expression, osteoblast maturation and bone formation 8,9 . Some transcription factors, like runt-related transcription factor 2 (Runx2) and Osterix (Osx), are related with increases in mesenchymal stem cells under mechanical strain. Runx2 is described as a positive regulator of osteoblast differentiation that can upregulate the expression of bone matrix protein genes, including collagen, type I, alpha 1 (Col1a1), osteopontin (Opn), bone sialoprotein (Bsp) and osteocalcin (Ocn) 10 . Osx has been shown to act as a downstream factor of Runx2 in the osteoblast maturation and enhance the proliferation and osteogenic lineage commitment of the bone marrow stromal cells 11 . Previously, it has been shown that mechanical loading exercise reduces the risk of bone loss, while it increases bone mass and bone remodeling processes 12,13 , bringing benefits to the musculoskeletal system of postmenopausal women 2 and female rodents 3,6 . However, there is little knowledge about mechanisms by which exercise reduces or reverses bone loss in aged females. In view of the well-known role of mesenchymal stromal cells in osteogenesis, a better Proliferative rate and alkaline phosphatase activity in BMSCs. The proliferative rate assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay and alkaline phosphatase (ALP) activity were analyzed in BMSCs after 0, 7, 14 and 17 days of osteogenic differentiation. A time-dependent increase in proliferation rate was observed in all experimental groups, indicating the ability to maintain proliferation during long-term culture. The effect of the donor's age on MTT reduction in BMSCs was only observed at 17 days when the culture from the aged rat cells eventually showed larger reduction (p = 0.026) in MTT into formazan crystals compared to the adult group, due the differentiation process of the BMSCs (Fig. 2a). However, physical activity did not alter this parameter because there was no significant difference between the cells from exercised aged and non-exercised aged animals (Fig. 2a).
The ALP activity (Fig. 2b) presented an increasing trend until a peak at day 14, with further decreasing trend, for all experimental groups, which is a characteristic pattern for this culture model. Regarding the differences between experimental groups, ALP activity was significantly lower in the aged groups, compared to the adult group at day 7 (p < 0.001), at day 14 (p < 0.001) and at day 17 (p < 0.001) (Fig. 2b). However, ST was able to generate a significant increase in the ALP activity rate in the exercised aged group, compared to the non-exercised aged group at day 7 (p = 0.004), and at day 17 (p = 0.018) (Fig. 2b).

Mineralization in BMSCs.
To verify whether ST-induced increase in ALP activity was accompanied by improved mineralization, we assessed the Alizarin red staining at 21 days (Fig. 2c). Alizarin Red staining resulted in marked differences between adult and aged groups (p < 0.001) (Fig. 2c, non-exercised aged group, the quantity of calcium deposition in the exercised aged group was significantly higher (p < 0.001), showing a 21% increase in mineralization (Fig. 2c,d).
Effect of ST practice in the osteogenic and adipogenic differentiation gene expression. We examined the transcription factors involved in the osteoblast differentiation gene expression at a time interval of 14 days. Runx2 expression was considerably increased in the adult group compared with the aged group (p = 0.040) (Fig. 3a,b). However, ST performed in the aging rats has provided an increase in the levels of Runx2 (p < 0.001) (Fig. 3a) and Osx (p = 0.035) (Fig. 3b), when compared with the aged group. Strength training effect on areal bone mineral density (aBMD) measured by dual-energy X-ray absorptiometry (a), maximum load (b), elastic modulus (c) and energy to maximum load (d) measured by compression test in femurs from adult and aged female rats. Images of the cross-sectional area were photographed (e), and cross-sectional area was measured by ImageJ software in femurs from adult and aged female rats (f). Data were expressed as means ± standard error (SEM) and examined using the unpaired Student's t-test. *p < 0.05 vs. adult, **p < 0.05 vs. aged rats. We also examined the peroxisome proliferator-activated receptor gamma (Pparγ) transcription factor involved in the adipogenesis differentiation gene expression, accomplished at a time interval of 14 days. Pparγ expression was significantly increased in the non-exercised aged group compared with the adult (p = 0.006) and exercised aged groups (p = 0.017) (Fig. 3d). At the 21 st day, the cells were stained with Oil Red O solution to identify lipid droplets. This staining revealed that the lipid accumulation in BMSCs was higher in the non-exercised aged group compared with the adult and exercised aged groups (Fig. 3c), contributing to the results obtained by Pparγ expression (Fig. 3d).

ST and extracellular matrix proteins expression.
To investigate the transcription factors involved in quality of extracellular matrix proteins, quantitative real-time PCR (qRT-PCR) analysis was performed. Bone morphogenic protein 2 (Bmp2) expression was significantly higher in the exercised aged compared with the non-exercised aged group (p < 0.001) (Fig. 4a), as well as for the Bsp expression (p = 0.027) (Fig. 4b). However, the aged group showed a considerable increase in Opn expression when compared with the adult group (Fig. 4c) demonstrating a delay in expression of osteogenic markers in the aged group. The results also indicated that ST increased the Ocn expression when compared with the aged group (p = 0.021) (Fig. 4d).
The production of pro-inflammatory cytokines after ST. The production of tumor necrosis factor alpha (TNF-α ) (Fig. 5a) and interleukin-6 (IL-6) (Fig. 5b) in the BMSCs supernatant, evaluated by ELISA assay, was significantly higher in the aged group when compared with the adult group (p = 0.038 and p = 0.017). However, the exercised aged groups showed decreased production of TNF-α in comparison with the non-exercised aged groups (Fig. 5a). We also observed a decline of IL-6 in these animals, although it was not significant (Fig. 5b).

Figure 2. MTT and ALP after strength training in aging.
Strength training effect on level of 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction (a) and alkaline phosphatase (ALP) activity (b) in the bone marrow mesenchymal stromal cells (BMSCs) from adult and aged female rats after osteogenic induction. Results were presented as values multiplied by one hundred. Calcium deposits by alizarin red (c) and alizarin red measured (d) on day 21 after osteogenic induction. Data were expressed as means ± standard error (SEM). Statistical analysis of data from MTT and ALP assays were done using one-way repeated measures ANOVA followed by Tukey's post-hoc test, to compare the differences across test days ( a p < 0.05 vs. 0 day in the same group, b p < 0.05 vs. 7 day in the same group), while the one-way ANOVA, followed by Bonferroni's post-hoc test, was used to compare the differences between the groups in the same day ( # p < 0.05 vs. adult in the same day and † p < 0.05 vs. aged in the same day). The results from alizarin red measurement were analyzed statistically using Student's t-test (*p < 0.05 vs. adult, **p < 0.05 vs. aged rats).

Discussion
Our results evidenced that the progressive load exercise program had a stimulatory effect in the bone of aged female rats. Our observations might be explained, at least in part, by an increase in both osteoblastic commitment and bone marrow mesenchymal stromal cells differentiation, effects that were probably due to the enhanced expression of transcription factors and matrix proteins that are important for bone metabolism. This study presents interesting results about the long-lasting effects of physical exercise during aging on biological mechanisms at the cellular level and the mechanical properties in bone during the periestropause period, when changes in bone homeostasis are observed, resulting in major probability of fractures by fragility.
In the present study, we demonstrated that progressive overload imposed in aged rats during ST significantly increased aBMD and was positively associated with maintained bone homeostasis. The mechanical integrity of bone, determined in this study by analysis of the maximum load, elastic modulus and energy to maximum load, showed that bone deformation threshold provided by the training program favored adaptations for strength and resistance, given the increase in aBMD and cross-sectional bone area from exercised aged rat. Furthermore, it is likely that the expression of growth factors and the increase of bone matrix production favored osteogenesis, consequently improving bone structural and mechanical properties in exercised aged animals. We believe that these findings reflect the intensity of the ST program, i.e., using low repetition/high overload 3,13,14 , in which aged rats displayed a MVCC value, expressed as relative value to the body weight, of 128.81% in comparison to the values obtained previous to ST program practice (Table 1). In addition, it is important to consider that the length of the training period (16 weeks) contributed to the significant differences observed in biomechanical properties. This means that even when applying high impact/high intensity programs, exercise frequency and its maintenance play a key role in bone adaptation 15 , such as demonstrated in this study.
Studies have shown that the osteogenic differentiation potential of cultured BMSCs changes according with the age of the donor and the treatment applied both in vivo and in vitro, by means of cellular subculture 6,7 . Others previous reports have shown that BMSCs cultures subjected to vibrations exhibited an increase in their intrinsic   potential for cellular activation and osteoblastic differentiation compared with those that were not vibrated 8 . Therefore, in order to obtain a cell population with a characteristic stromal microenvironment phenotype, we used the primary culture of BMCSs from aged rats that were subjected to ST compared with primary culture from aged rats did not undergo ST. We analyzed whether ST practice regulated biological mechanisms at the cellular level in aged rats. BMSCs in vitro should present a characteristic phenotype, once again because the pool of cells present in bone marrow are mesenchymal stem cells 16 directly involved in stromal environment maturation 17 . This study showed that the biological characteristics of BMSCs exhibited sustained continuous growth and proliferation ability, which was maintained during long-term culture. Although cell proliferation was observed during the osteogenesis in all experimental groups, osteoblast phenotype maturation signals, like up regulation of ALP, an essential marker for matrix mineralization 18 , were lower in the aged groups, the observed. Nevertheless, the exercised aged group presented higher ALP activity when compared to the non-exercised aged group, reflecting the extracellular matrix maturation process 19 under the influence of ST. These markers have been described to decrease with donor age, and this has been established both in human mesenchymal stem cells (hMSC) isolated from the femoral heads of aged patients 20 and the mesenchymal stem cell isolated from rats of various ages 21 . Nevertheless, in our data, ST practice was able to promote an increase in the differentiation of BMSCs derived from aged rat into osteoblasts, resulting in a greater biological mineralization, reflecting the potential advanced cell differentiation 22 .
A study conducted by Hell et al. 6 with young and adult female rats demonstrated an increase in the osteogenic differentiation of BMSCs isolated from active adult rats. The difference between the previous study and ours is that we studied animals with mature skeletons 23 and with changes in the estrous cycle, which features an appropriate animal model for studying bone loss linked to aging. In female rats, the incidence of regular estrous cyclicity decreases progressively during aging and their estrous cycles tend to become irregular, usually with prolonged estrous and diestrous, characterizing periestropause 24,25 , and similar to perimenopause in women 26,27 , they have an increased risk of fragility fractures in this period 2 . Therefore, until recently, the effect of physical activity, specifically ST, in the osteogenic differentiation of BMSCs cultures from aged donor rats, had not been previously demonstrated.
Up to now, these findings indicate that mechanical stimulus can reverse the functional decline in the cellular microenvironment of cultured BMSCs from aged donors, resulting in increased bone formation by greater differentiation of the BMSCs into the osteoblast lineage in vitro. Subsequently, it was observed an increase in the expression of osteoblast-specific transcription factors, Runx2 and Osx promoted by ST practice. These results suggest that ST-induced mechanical stimulus on bone drive the differentiation of mechanosensitive BMSCs. This results in a mechanical activation of bone cells that changes the bone canalicular fluid gradient and increases intracellular calcium concentration, in addition to osteogenic factors and bone matrix production 12,13 . The evaluation of Pparγ expression, a key regulator of adipocyte differentiation, confirmed that the mechanical stimulus directly increased the differentiation and bone formation and adversely altered the marrow adiposity, confirmed by decreased expression of Pparγ in the BMSCs of aged rats that performed ST. In 'this study, the exercised aged group presented higher osteogenic potential due to an increased expression of Runx2 and Osx, leading to the differentiation of BMSCs away from adipocytes and towards osteoblasts.
These results highlight the importance of the Runx2 and Osx in directly stimulating the transcription of important osteoblast-associated proteins that support proliferation, matrix formation and mineralization 16 , such as Bmp2, Bsp, Opn, and Ocn 10,11 . Thus, our results showed that the bone responds to mechanical stimulus with increased expression of matrix proteins, such as Bmp2 and Bsp, both necessary for the initiation of bone mineralization 28 . The expression of the non-collagenous protein Opn is necessary to initiate crystal formation and prevent the premature precipitation of calcium phosphate crystals that do not have the well-coordinated structure of hydroxyapatite 28,29 . Moreover, Opn may facilitate the binding of osteoclasts, favoring bone resorption 28 . Thus, considering that osteoclast activity increases with age 30 , the increased expression of Opn in the aged group, suggests a prevalence of the resorptive phenotype. Another protein, Ocn, which acts as a chemo-attractant for the recruitment and differentiation of the osteoclast progenitors and regulates the quantity of mineral deposition 28 , was significantly expressed in the exercised aged group. These results show that ST acts positively in bone modulation even under the influence of aging, when there is a poor quality of extracellular matrix due to an increase in bone resorption in comparison to bone formation 30 .
As a final point, we evaluated pro-inflammatory cytokine levels, such as TNFα and IL-6, which present negative effects on osteoblast differentiation, favoring elevated bone-resorbing activity 5 . In this study, we evaluated the physiological aging female rat to study primary osteoporosis. In women, osteoporosis, most often, is related with estrogen reduction, similar to what happens in aged female rats 3,31 . As the levels of estrogen decrease, the production of TNFα and IL-6 increase and play an important role in the pathogenesis of inflammatory bone loss through stimulation of bone resorption and inhibition of osteoblastic bone formation 32 . In this study, the exercises performed by aged female rats stimulated osteogenic potential by increasing the expression of osteoblast-specific transcription factors, decreasing expression of Pparγ, and decreasing the production of TNF-α . These results show that ST triggers a series of physiological responses involving changes in the bone microenvironment, benefiting biomechanical parameters of bone tissue in aged female rats.
Taken together, the in vivo and in vitro analyses demonstrated the positive effects of exercise during aging. We found that ST improves mechanical competence of the bone in vivo in exercised aged rats by modulating processes such as differentiation and up regulation of osteoblast phenotypic marker genes, while simultaneously suppressing adipocyte expression and pro-inflammatory cytokine production. These processes promote biological mineralization in vitro. These results add new information about molecular and cellular mechanisms underlying the osteogenic differentiation in the aging and provide good basis for preclinical studies.
Scientific RepoRts | 7:42878 | DOI: 10.1038/srep42878 Methods Animals. Animal procedures were approved by Ethics Committee for Research Involving Animals of the University Estadual Paulista (CEUA: 00462/2012) and complied with the Guide for Care and Use of Laboratory Animals. Multiparous female rats (Rattus norvergicus albinus) at ages 5 (adult group) and 17 months old (aged group), were housed in a controlled room. Estrous cycle was checked through vaginal smears taken daily to confirm regularity in the cycle of the adult rats, and irregularity in the cycle of the aged females, characterizing the periestropause period 24,25,27 . Only adult animals (n = 10) with regular cycles and aged animals (n = 20) with irregular cycles were included in this study. Aged rats were randomly allocated into two groups: aged animals (n = 10) and exercised aged animals (n = 10). The ST in exercised aged group was carried out by climbing sessions on a ladder during 16 weeks. Adult animals were euthanized at 9 months old, and aged animals at 21 months old, always seventy-two hours after the last ST session. Adult rat group was used as a reference to compare with aged rat, since a peak in bone mass occurs around 9th month of life 23 . Both femurs from all experimental groups were quickly collected for further analysis.
Strength training protocol and MVCC. Aged rats completed the ST program 33 , by performing climbing sessions on a ladder (1.1 × 0.18 m, 2-cm grid, 80° incline -engineered by department of maintenance Unesp, SP, Brazil). Animals underwent 1 week of acclimation performed without overload. Seventy-two hours after the last acclimated session, the initial MVCC of each animal was obtained. A load apparatus (Plastic tube, BD Biosciences ® , MA, USA) containing steel balls (Cabana S/A, SP, Brazil) corresponding to 75% of the body mass was secured to the proximal portion of the rat's tail with a self-adhesive foam strip (Missner & Missner Ltda, SC, Brazil). The rats performed repeated climbs with the load apparatus, resting 5 min between each climb. Then, 30 g of load was added to the load apparatus, and a new repetition was performed until climbing failure. The value of the load before climbing failure was considered the final MVCC. The test was performed for 15 days in a row, so that the correct load was applied in each animal. ST protocol consisted of 3 sessions per week (nonconsecutive days) for 16 weeks. Each session was composed of 6 sets of climbs, containing 48 and 62 dynamic movements (isotonic) per series. The first week of the ST protocol began with an overload corresponding to 60% of the MVCC, which was increased to 70% of the MVCC in the second week, and finally to 80% of the MVCC in the third week, and this last established load was maintained until the end of the protocol.
Bone mineral density measurements. aBMD was measured in left femurs (10 specimens in each group) by DEXA (Lunar DPX Alpha, WI, USA). The equipment was calibrated according to manufacturer instructions, and the device software was used for measuring BMD in small animals. aBMD was calculated by dividing bone mineral content by bone area (cm 2 ) using the software associated with the DEXA scanner in order to determine the aBMD (g/cm 2 ).
Biomechanical compression testing. Biomechanical properties of the left femurs were assessed by the compression test (Universal Testing Machine; DL 3000, EMIC ® , PR, Brazil). All femurs were cut in the proximal third of the diaphysis (measuring 6 mm in length) using a diamond cut-off wheel (Dremel ® 540; IL, USA) coupled to a portable low speed motor. The bone cortical wall thickness of the samples was photographed and processed with ImageJ software (NIH, Maryland, USA), version 1.48 v, for Windows. Cross-sectional area values were acquired by obtaining the ratio between bone length (mm) and diameter (mm 2 ). The biomechanical compression test was performed with a deformation rate of 5 mm/min with standardized parameters of loaded cells to 2000 N of capacity until the bone fractured 34 . The machine crossbar load and displacement was monitored and recorded using the device software. A load versus deformation graph was created from the data and the values for maximum load (N) were obtained. The elastic modulus (MPa) and energy to maximum load (mJ) were obtained from the tension and elongation curves, by using the cross-sectional area and initial length of the samples.
Isolation and primary culture of bone marrow-derived mesenchymal stem cells. Primary culture of BMSC was established from rights femurs, using a protocol adapted from elsewhere 35 . Bone marrow (10 specimens in each group) was flushed out using 20 mL of minimal essential medium (MEM), containing 10% fetal bovine serum (FBS), 2 mM l-glutamine, and 1% antibiotics (100 U/mL penicillin G, 100 μ g/mL streptomycin and 0.3 μ g/mL amphotericin B) (Sigma Aldrich ™ , St. Louis, MO, USA). After centrifugation, the cell suspension was filtered through a 70-μ m size cell strainer and through 22 and 26-gauge needles and then seeded in wells of 24-wellplates (TPP, Switzerland) at a density of 19 × 10 5 cells/cm 2 and cultured at 37 °C under a 5% CO 2 95% air atmosphere. After 10 days, under subconfluence (80%), the cells were cultured in a proliferation medium (MEM) or osteogenic medium (MEM supplemented with 10 nM β -glycerophosphate, 50 μ g/mL ascorbic acid and 10 nM dexamethasone), and subjected to experimental tests after 0, 7, 14, 17 and 21 days in culture, after the addition of osteogenic medium.
MTT assay for cell proliferation. Cell proliferation and viability were measured by the MTT reduction assay after 0, 7, 14 and 17 days 36 in culture. MTT reagent (Sigma-Aldrich ™ ) was added to each sample to allow the formation of MTT formazan. The resulting formazan was reduced with ethanol (Merck, Darmstadt, Germany), and measured at 570 nm by a spectrophotometric microplate reader (Molecular Devices' , CA, USA). Cell proliferation was determined by comparing the absorbance of samples to a standard curve and the results were presented with the original values multiplied by one hundred.
Alkaline phosphatase activity. ALP activity was determined after 0, 7, 14 and 17 days 37 in culture using a commercial kit (Labtest Diagnóstica, MG, BRA). Enzyme activity was detected by thymolphthalein release and measured at 590 nm using a spectrophotometric microplate reader (Molecular Devices'). The specific activity was Scientific RepoRts | 7:42878 | DOI: 10.1038/srep42878 calculated by enzyme activity normalized by total protein content 38 . Results were presented multiplied by one hundred.
Alizarin red staining. Mineralization in osteoblast cultures was determined by Alizarin Red S staining 39 after 21 days in culture. Briefly, cells were fixed with paraformaldehyde (Merck) at 10% for 30 min, washed with distilled water, and stained with 20 mg/mL Alizarin Red S (Sigma Aldrich ™ ) for 30 min. Stained cells were digitalized using a high resolution scanner and staining was quantified by spectrophotometric measure at 405 nm using a microplate reader (Molecular Devices').
Oil red O staining. Cells were stained for fat vacuoles by using oil red O (Sigma Aldrich ™ ) staining after 21 days 40 in culture. Cells were rinsed in PBS, fixed in 10% formaldehyde (Merck), stained in 0.3% oil red O solution (Sigma Aldrich ™ ) and the lipid droplets of differentiated cells were then obtained from each experimental group.
Images were captured using a digital camera system (Olympus DP12-2) coupled to an inverted optical microscope (Olympus CKX41-Olympus Optical CO., Ltd.; Japan).
Total RNA isolation and gene expression. Total RNA from biological samples were isolated using Trizol reagent (Invitrogen, Life Technologies, NY, USA) after 14 days in culture. Total RNA from each sample was treated with DNAse I and reverse transcribed to complementary DNA (cDNA) using SuperScript ™ II Reverse Transcriptase (Invitrogen). Expression of target genes was determined by qRT-PCR using gene-specific TaqMan probes (Applied Biosystems/Life Technologies, Paisley, UK) with Taqman ® Gene Expression Master Mix on the StepOne ™ Real-Time PCR System (Applied Biosystems/Life Technologies). Probes used for RT-PCR are listed: Ocn (Rn00566386_g1); Opn (Rn00563571_m1); Bsp (Rn00561414_m1); Runx2 (Rn01512298_ml); Osx (Rn02769744_s1); Pparg (Rn00440945_m1) and Bmp2 (Rn90931). The relative amount of each transcript was determined using the delta cycle threshold (Δ Ct) values as previously described 41 . Changes in gene expression were calculated using relative quantitation of a target gene against endogenous, unchanged Actb (Rn00667869_m1) standard. Samples from BMSCs adult rats were used as a calibrator.
Measurement of TNF-α and IL-6 production. Production of TNF-α and IL-6 was evaluated with ELISA. Briefly, supernatant medium of BMSCs was collected after 14 days in culture, centrifugated and measured using ELISA Kit to TNF-α (R&D Systems, Minneapolis, USA) and an IL-6 (R&D). Concentration of each TNF-α and IL-6 was calculated from a standard curve in pg/mL. Statistical analysis. Data were analyzed with Graph Pad Prism ® software (CA, USA), version 6.0, for Windows. Values were presented as mean ± standard error (SEM) and significance of differences was assessed using the unpaired Student's t-test. Significance level was set at 5% (p < 0.05) for comparisons between adult vs. aged; aged vs. exercised aged rats. Statistical analysis of data from MTT and ALP assays were done using repeated measures one-way ANOVA, followed by Tukey's post-hoc test, to compare the differences across test days, while the one-way ANOVA, followed by Bonferroni's post-hoc test, was used to compare the differences between the groups in the same day.